SADII RESEARCH PROJECT 3: Genetic and hormonal mechanisms of sex differences in immune responses and influenza vaccine efficacy in young and aged mice SUMMARY Using C57BL/6 mice and an inactivated 2009 H1N1 vaccine prime and boost strategy, we have shown that greater antibody in young adult females is sufficient for protection against influenza. Following influenza virus vaccination, young adult female mice have greater antibody responses and protection against challenge with an influenza drift variant virus than males. Antibody derived from vaccinated females is better at protecting both nave males and females than antibody derived from males, and this protection is associated with increased antibody specificity and avidity to the influenza virus. We have identified two factors that reduce female-biased antibody responses and protection following influenza vaccination?old age and deletion of genes that control somatic hypermutation (activation-induced cytidine deaminase [Aicda]) and antibody class switching recombination in B cells (toll like receptor 7 [Tlr7]). Because secretion of sex steroids is reduced to a greater extent in females than males with older age, the age-associated reduction in female-biased immunity suggests a role for sex steroids. In contrast, because Tlr7 is on the X chromosome and escapes X inactivation in B cells, female-biased immunity to influenza may also reflect direct effects sex chromosomes. The overarching goal of SADII Research Project 3 is to analyze and manipulate the biological factors mediating the sex and age differences in immunity to influenza, through completion of three aims: Aim 1 will systematically evaluate how sex chromosome complement either directly through imbalanced expression of X and Y genes or indirectly via sex hormone concentrations cause sex differences in immunity and protection from influenza, using transgenic mice (i.e., Four Core Genotype); Aim 2 will tease apart the impact of chronological age versus reproductive senescence on reduced sex differences in humoral immunity and protection from influenza in aged as compared with young adult mice; and Aim 3 will characterize sex differences in transcriptional activity and B-cell influenza-specific receptor repertoire following vaccination, focusing initially on activation of pathways that we have shown to be differentially regulated between the sexes (e.g., TLR7 signaling) in mice, as well as novel pathways that are uncovered in the analyses of cells from Projects 1 and 2. Together, the studies in Research Project 3 will provide translational mechanistic insights that can be used test hypotheses derived from the human data from Research Projects 1 and 2.